Methods for increasing polypeptide production

ABSTRACT

The invention provides methods of increasing the production of polypeptides, optionally recombinant polypeptides, from mammalian cells using xanthine derivatives or hybrid polar compounds and cultures containing the same. Combinations of inducers including a hybrid polar compound and/or a xanthine derivative and/or an alkanoic acid can also be used, optionally at temperatures less than 37° C.

This application is a continuation of U.S. application Ser. No.10/400,334, filed Mar. 27, 2003, which claims the benefit of U.S.Provisional Application Nos. 60/368,246 and 60/368,248, both filed Mar.27, 2002, all of which are incorporated herein in their entirety.

FIELD OF THE INVENTION

The invention is in the field of polypeptide production, particularlyrecombinant polypeptide production in cell culture.

BACKGROUND

Polypeptides are useful in a variety of diagnostic, therapeutic,agricultural, nutritional, and research applications. Althoughpolypeptides can be isolated from natural sources, the isolation oflarge quantities of a specific polypeptide from natural sources may beexpensive. Also, the polypeptide may not be of uniform quality due tovariation in the source material. Recombinant DNA technology allows moreuniform and cost-effective large-scale production of specificpolypeptides.

One goal of recombinant polypeptide production is the optimization ofculture conditions so as to obtain the greatest possible productivity.Incremental increases in productivity can be economically significant.Some of the methods to increase productivity in cell culture includeusing enriched medium, monitoring osmolarity during production,decreasing temperatures during specific phases of a cell culture, and/orthe addition of sodium butyrate (see, e.g., U.S. Pat. No. 5,705,364).

However, as more polypeptide-based drugs demonstrate clinicaleffectiveness and increased commercial quantities are needed, availableculture facilities become limited. Accordingly, there remains a need inthe art to continually improve yields of recombinant polypeptides fromeach cell culture run.

SUMMARY

As shown by the experimental data reported herein, xanthine derivativesand/or hybrid polar compounds can dramatically induce the production ofpolypeptides, especially recombinant polypeptides, from mammalian celllines. Moreover, xanthine derivatives and/or hybrid polar compounds canbe used in combination with other induction methods to further increasepolypeptide expression.

Thus, in one aspect, the invention provides a method for producing apolypeptide, which may be a recombinant polypeptide, comprisingculturing a mammalian cell line in a growth phase followed by aproduction or induction phase, which can occur at a temperature of lessthan 37° C., and adding to the culture during the production phase axanthine derivative. The addition of the xanthine derivative canincrease the production of the polypeptide. The mammalian cell line canbe a cell line that has been genetically engineered to produce thepolypeptide or a hybridoma cell line that can produce an antibody. Thexanthine derivative may be caffeine at a concentration from about 0.01millimolar to about 5.0 millimolar or from about 0.01 millimolar toabout 3.0 millimolar. In some embodiments, the mammalian cell line is aCHO cell line, and it may have been transformed with a recombinantvector encoding the recombinant polypeptide. Optionally, the vector cancomprise a cytomegalovirus (CMV) promoter. Typically, the cell does notnaturally express the polypeptide or only naturally expresses thepolypeptide at very low levels (in the absence of genetic engineering).The polypeptide may be a recombinant fusion polypeptide or a human orhumanized antibody. The production or induction phase can occur at atemperature from about 29° C. to about 36° C. or from about 30° C. toabout 33° C. The growth phase can occur at a temperature from about 35°C. to about 38° C.

Optionally, at least two different xanthine derivatives can be added.The xanthine derivative(s) can be selected from the group consisting ofcaffeine, 3-isobutyl-1-methylxanthine, theophylline, theobromine,pentoxyphylline, and aminophylline or from a subset of this group. Iftwo different xanthine derivatives are added, they can be caffeine and3-isobutyl-1-methylxanthine. Xanthine derivatives can be added multipletimes during the culturing of the cell line, and the cell line can becultured in the presence of the xanthine derivative for at least about 5days. The concentration of each xanthine derivative added to the culturecan be from about 0.001 millimolar to about 3 millimolar. Therecombinant polypeptide can be collected from the medium and formulated.The medium may further comprise a hybrid polar compound and/or analkanoic acid. The hybrid polar compound can be hexamethylenebisacetamide, optionally at a concentration from about 0.1 millimolar toabout 5 millimolar. The xanthine derivative can be caffeine, optionallyat a concentration from about 0.1 millimolar to about 4 millimolar. Thealkanoic acid can be a salt of butyric acid, optionally at aconcentration from about 0.1 millimolar to about 2 millimolar. Themammalian cells can be cultured at a temperature from about 29° C. toabout 36° C. or from about 30° C. to about 33° C. The mammalian cellscan be cultured in a growth phase at a first temperature from about 35°C. to about 38° C. before they are shifted to a production phase at asecond temperature from about 29° C. to about 36° C., wherein the secondtemperature can be lower than the first temperature. The xanthine can beadded at the time of the shift from the first temperature to the secondtemperature and/or before and/or after the shift.

In another aspect the invention provides a method for producing arecombinant polypeptide comprising growing in culture a mammalian cellline, optionally a CHO cell line that has been genetically engineered toproduce the recombinant polypeptide, and adding to the culture medium atleast one xanthine derivative selected from the group consisting oftheobromine and caffeine. The addition of the xanthine derivative canincrease the production of the recombinant polypeptide. The mammaliancell line may have been transformed with a recombinant vector encodingthe recombinant polypeptide. Optionally, the vector can comprise acytomegalovirus (CMV) promoter. Typically, the cell line does notnaturally express the recombinant polypeptide or only naturallyexpresses the recombinant polypeptide at very low levels (in the absenceof genetic engineering). The recombinant polypeptide may be arecombinant fusion polypeptide or a human or humanized antibody. Thecell line can be cultured in a growth phase, which is distinct from aproduction or induction phase. The production phase can occur at atemperature less than 37° C. The cell line can be cultured at atemperature of from about 29° C. to about 36° C. or from about 30° C. toabout 33° C. Optionally, at least two different xanthine derivatives canbe added. Xanthine derivatives can be added multiple times during theculturing of the cell line. The concentration of each xanthinederivative added to the culture can be from about 0.001 millimolar toabout 3 millimolar. The recombinant polypeptide can be collected fromthe medium and formulated. The mammalian cell line can be cultured at afirst temperature from about 35° C. to about 38° C. before it is shiftedto a second temperature from about 29° C. to about 36° C., and thexanthine derivative can be added at the time of the shift from the firsttemperature to the second temperature and/or before and/or after theshift. The second temperature can be lower than the first temperature.

In another aspect, the invention provides a culture comprising a CHOcell genetically engineered to produce a polypeptide, a productionmedium, and at least one xanthine derivative selected from the groupconsisting of caffeine, 3-isobutyl-1-methylxanthine, theophylline,theobromine, pentoxyphylline, and aminophylline or from a subset of thisgroup. The culture can comprise at least two xanthine derivatives. Theconcentration of each xanthine derivative present can be from about0.001 millimolar to about 3 millimolar or from about 0.01 millimolar toabout 3 millimolar. The culture can comprise serum-free medium, and maycomprise no added protein or may comprise insulin or IGF-1.Additionally, the culture can comprise dimethylformamide,dimethylsulfoxide, or dimethylacetamide. The invention, because of itslow cost and convenience, is particularly useful for large scaleculturing of CHO cells. The culture can be a large scale culture of atleast 100 liters, or even at least 500 liters, in size. The culture cancomprise a homogeneous CHO cell line.

In still another aspect, the invention encompasses a culture comprisinga CHO cell genetically engineered to produce a polypeptide, a productionmedium, and at least one xanthine derivative, wherein the culture isgrown at less than 37° C. for at least part of its life. The xanthinederivative or derivatives present can be within the concentration rangefrom about 0.001 millimolar to about 3 millimolar or from about 0.01millimolar to about 3 millimolar, and the culture can contain at leasttwo different xanthine derivatives. The xanthine derivatives can beselected from the group consisting of caffeine,3-isobutyl-1-methylxanthine, theophylline, theobromine, pentoxyphylline,and aminophylline or from a subset of this group such as caffeine,theobromine and pentoxyphylline. The size of the culture can be at least100 liters, and the production medium can be serum-free medium and cancomprise either no added protein or insulin or IGF-1. The culture cancomprise a homogeneous CHO cell line.

In a further aspect, the invention includes a method for producing apolypeptide in a culture of mammalian cells comprising incubating theculture at a temperature of about 37° C. and thereafter incubating theculture at a temperature from about 29° C. to 36° C., and adding to theculture a xanthine derivative during the incubation at a temperaturefrom about 29° C. to 36° C., wherein the polypeptide is a recombinantpolypeptide or an antibody. The xanthine derivative can be selected fromthe group consisting of caffeine, 3-isobutyl-1-methylxanthine,theophylline, theobromine, pentoxyphylline, and aminophylline or from asubset of this group such as caffeine, theobromine, and pentoxyphylline.The mammalian cells can be hybridoma cells or CHO cells. The xanthinederivative or derivatives present can be within the concentration rangefrom about 0.001 millimolar to about 3 millimolar or from about 0.01millimolar to about 3 millimolar, and the culture can contain at leasttwo different xanthine derivatives. Xanthine derivatives can be addedmultiple times during the culturing of the cell line.

The invention provides a method for producing a recombinant polypeptidecomprising culturing a mammalian cell line, in some embodiments a CHOcell line, at a temperature from about 29° C. to about 36° C.,optionally at temperatures between about 29° C. and 35° C. or from about30° C. to about 33° C., in a medium comprising a hybrid polar compound.The medium can be serum free. The addition of the hybrid polar compoundcan increase the production of the recombinant polypeptide. The hybridpolar compound can be hexamethylene bisacetamide, optionally at aconcentration from about 0.1 millimolar to about 20 millimolar or fromabout 0.1 millimolar to about 5 millimolar. Furthermore, the medium maycomprise an alkanoic acid, such as a salt of butyric acid, at aconcentration, for example, from about 0.05 millimolar to about 10millimolar, optionally from about 0.1 millimolar to about 2 millimolar.Furthermore, the medium may comprise a xanthine derivative, for example,caffeine, at a concentration from about 0.005 millimolar to 10millimolar, optionally from about 0.01 millimolar to 4 millimolar orfrom about 0.1 millimolar to 4 millimolar. The mammalian cells can becultured at a first temperature from about 35° C. to about 38° C. beforethey are shifted to a second temperature between about 29° C. and 36°C., and the hybrid polar compound can be added after the shift from thefirst temperature to the second temperature. The mammalian cells may begenetically engineered to produce a polypeptide, optionally a secretedpolypeptide that can be recovered from the medium, including RANK:Fc,type II interleukin-1 receptor, TNFR:Fc, CD40 ligand, TRAIL,flt3-ligand, IL-4 receptor, G-CSF, erythropoietin, an antibody, or asubstantially similar polypeptide, among others.

In another embodiment, the invention provides an improved method forproducing a polypeptide by culturing mammalian cells comprisingculturing the cells in a medium comprising a hybrid polar compound,optionally at temperatures from about 29° C. to about 36° C., betweenabout 29° C. and 35° C., or from about 30° C. to about 33° C. The hybridpolar compound may be hexamethylene bisacetamide, optionally at aconcentration between about 0.1 millimolar and about 5 millimolar. Theaddition of the hybrid polar compound can increase the production of thepolypeptide, which may be a recombinant polypeptide. Furthermore, themedium may comprise an alkanoic acid, for example, butyric acid,optionally at a concentration from about 0.05 millimolar to about 10millimolar or from about 0.1 millimolar to about 2 millimolar.Furthermore, the medium may comprise a xanthine, such as, for example,caffeine, optionally at a concentration from about 0.005 millimolar to10 millimolar or from about 0.01 millimolar to 5 millimolar. Optionally,the polypeptide may be RANK:Fc, type II interleukin-1 receptor, TNFR:Fc,CD40 ligand, TRAIL, flt3-ligand, IL4 receptor, GM-CSF, erythropoietin,an antibody, or a substantially similar polypeptide among others.

In another aspect, the invention provides a method for obtaining apolypeptide, optionally a recombinant polypeptide, comprising recoveringthe polypeptide from medium in which mammalian cells have been grown,wherein the mammalian cells can secrete the polypeptide and are grown attemperatures between about 29° C. and 35° C., optionally from about fromabout 30° C. to about 33° C., in medium comprising hexamethylenebisacetamide. The hexamethylene bisacetamide may be present atconcentrations between about 0.1 millimolar and about 5 millimolar.Furthermore, the medium may comprise an alkanoic acid, for example,butyric acid, optionally at a concentration from about 0.05 millimolarto about 10 millimolar or from about 0.1 millimolar to about 2millimolar. Furthermore, the medium may comprise a xanthine, forexample, caffeine, optionally at a concentration from about 0.005millimolar to 10 millimolar or from about about 0.01 millimolar to 5millimolar. The polypeptide may be RANK:Fc, type II interleukin-1receptor, TNFR:Fc, CD40 ligand, TRAIL, flt3-ligand, IL-4 receptor,G-CSF, erythropoietin, an antibody, or a substantially similarpolypeptide, among others.

In a further embodiment, the invention comprises method for producing arecombinant polypeptide comprising culturing mammalian cells in a mediumcomprising a hybrid polar compound and a xanthine, wherein the mammaliancells have been genetically engineered to express the recombinantpolypeptide. The medium may further comprise an alkanoic acid, such as,for example, a salt of butyric acid, which may be at a concentrationfrom about 0.1 millimolar to about 2 millimolar. The hybrid polarcompound can be hexamethylene bisacetamide, which may be at aconcentration from about 0.1 millimolar to about 5 millimolar, and/orthe xanthine can be caffeine, which may be at a concentration from about0.1 millimolar to about 4 millimolar. The cells can be cultured at atemperature from about 29° C. to about 36° C. or from about 30° C. toabout 33° C. The mammalian cells can be cultured at a first temperaturefrom about 35° C. to about 38° C. before they are shifted to a secondtemperature from about 29° C. to about 36° C., and the hybrid polarcompound and the xanthine can be added at the time of the shift from thefirst temperature to the second temperature and/or before and/or afterthe shift. The can be medium can be serum free.

In a further embodiment, the invention encompasses a method forproducing a polypeptide, optionally a recombinant polypeptide,comprising culturing mammalian cells in a medium comprising a hybridpolar compound and an alkanoic acid, wherein the mammalian cells mayhave been genetically engineered to express the recombinant polypeptide.The hybrid polar compound can be hexamethylene bisacetamide, and thehybrid polar compound can be present at a concentration of from about0.5 millimolar to about 10 millimolar or at a concentration betweenabout 0.5 millimolar and 2.5 millimolar. The alkanoic acid can be a saltof butyric acid, and the alkanoic acid can be present at a concentrationfrom about 0.1 millimolar to about 5 millimolar or at a concentrationbetween about 0.1 millimolar and about 2.0 millimolar. The mammaliancells can be cultured at a temperature from about 29° C. to about 36°C., and the medium can be serum free. The mammalian cell line can becultured at a first temperature from about 35° C. to about 38° C. beforethey are shifted to a second temperature from about 29° C. to about 36°C., and the hybrid polar compound and the alkanoic acid may be addedafter the shift from the first temperature to the second temperature.The medium can further comprise a xanthine derivative at a concentrationfrom about 0.001 millimolar to about 5.0 millimolar. The mammalian cellline can be a hybridoma cell line or a CHO cell line.

In still another embodiment, the invention provides a method forproducing a polypeptide comprising culturing a mammalian cell line in aproduction phase at a second temperature from about 30° C. to 34° C. ina medium comprising a hybrid polar compound, wherein the productionphase follows a growth phase at a first temperature from about 35° C. toabout 38° C. The polypeptide can be a recombinant polypeptide or anantibody. The hybrid polar compound can be hexamethylene bisacetamide,optionally at a concentration from about 0.1 millimolar to about 5millimolar. The hybrid polar compound may be added after the shift fromthe first temperature to the second temperature. The medium can furthercomprise an alkanoic acid, which can be a salt of butyric acid,optionally at a concentration from about 0.05 millimolar to about 10.0millimolar. The medium can also comprise a xanthine derivative,optionally at a concentration from about 0.001 millimolar to about 5.0millimolar. The medium can be serum free. The mammalian cell line can bea hybridoma cell line or a CHO cell line.

The invention also provides a method for producing a polypeptidecomprising culturing a mammalian cell line in a medium comprising ahybrid polar compound at a concentration between about 0.5 millimolarand 2.5 millimolar, an alkanoic acid at a concentration from about 0.1millimolar and 2.0 millimolar, and a xanthine derivative at aconcentration from about 0.001 millimolar to about 4 millimolar.

In still another embodiment, the invention provides a method forproducing a polypeptide, optionally RANK:Fc, type II interleukin-1receptor, TNFR:Fc, CD40 ligand, TRAIL, flt3-ligand, IL4 receptor, G-CSF,erythropoietin, an antibody, or a substantially similar polypeptide,comprising culturing mammalian cells, which may have been geneticallyengineered to produce any of these polypeptides, in a medium comprisingbetween about 0.1 millimolar and about 5 millimolar HMBA, from about 0.1millimolar to about 2 millimolar butyric acid, and from about 0.1millimolar to about 4 millimolar caffeine at a temperature from about29° C. to about 36° C. or from about 30° C. to about 33° C.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the percentage of the total cells that are viable under theindicated conditions at 31° C. for the #9 CHO cell line as a function ofdays in culture.

FIG. 2 shows the micrograms of protein per milliliter of cell culture,i.e., protein titer, under the indicated conditions at 31° C. for the #9CHO cell line as a function of days in culture.

FIG. 3 shows the micrograms of protein per 10⁶ cells per day under theindicated conditions at 31° C. for #9 CHO cell line as a function ofdays in culture.

FIG. 4 shows a graph displaying the concentration of an antibody againstmurine IL4 receptor recovered from medium as a function of days ofgrowth of a CHO cell line comprising a vector encoding the antibody atthe stated temperatures in the presence or absence of HMBA or sodiumbutyrate. Markings are as follows: —▴—, no inducer 37° C.; —▪—, noinducer 34° C.; —▪—, 0.5 millimolar sodium butyrate 34° C.; —▪—, 2.0millimolar HMBA 34° C.; —●—, no inducer 31° C.; —●—, 0.5 millimolarsodium butyrate 31° C.; and —●—, 2.0 millimolar HMBA 31° C.

DETAILED DESCRIPTION OF THE INVENTION

An “antibody” is a polypeptide or complex of polypeptides, each of whichcomprises at least one variable antibody immunoglobulin domain and atleast one constant antibody immunoglobulin domain. Antibodies may besingle chain antibodies, dimeric antibodies, or some higher ordercomplex of polypeptides including, but not limited to, heterodimericantibodies. A “human antibody” is an antibody encoded by nucleic acidsthat are ultimately human in origin. Such an antibody can be expressedin a non-human cell or organism. For example, DNA encoding a humanantibody can be introduced into tissue culture cells and expressed intransformed cell lines. Alternatively, human antibodies can be expressedin transgenic animals such as, for example, the transgenic micedescribed in Mendez et al. ((1997), Nature Genetics 16(4): 146-56). Suchtransgenic mice are utilized in making the fully human antibodies inU.S. Pat. No. 6,235,883 B1. Human antibodies can also be expressed inhybridoma cells. A “humanized antibody” is a chimeric antibodycomprising complementarity determining regions (CDR1, CDR2, and CDR3)from a non-human source and other regions that conform to sequences inhuman antibodies (and may be of human origin) as explained in, e.g.,U.S. Pat. Nos. 5,558,864 and 5,693,761 and International PatentApplication WO 92/11018.

A “constant antibody immunoglobulin domain” is an immunoglobulin domainthat is identical to or substantially similar to a C_(L), C_(H)1,C_(H)2, C_(H)3, or C_(H)4, domain of human or animal origin. See e.g.Hasemann and Capra, Immunoglobulins: Structure and Function, in WilliamE. Paul, ed., Fundamental Immunology, Second Edition, 209, 210-218(1989); Kabat et al., Sequences of Proteins of Immunological Interest,U.S. Dept. of Health and Human Services (1991).

An “F_(C) portion of an antibody” includes human or animalimmunoglobulin domains C_(H)2 and C_(H)3 or immunoglobulin domainssubstantially similar to these. For discussion, see Hasemann and Capra,supra, at 212-213 and Kabat et al., supra.

Cells have been “genetically engineered” to express a specificpolypeptide when recombinant nucleic acid sequences that allowexpression of the polypeptide have been introduced into the cells usingmethods of “genetic engineering,” such as viral infection with arecombinant virus, transfection, transformation, or electroporation. Seee.g. Kaufman et al. (1990), Meth. Enzymol. 185: 487-511; CurrentProtocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, NewYork, 1988, and quarterly updates). Infection with an unaltered,naturally-occurring virus, such as, for example, hepatitis B virus,human immunodeficiency virus, adenovirus, etc., does not constitutegenetic engineering as meant herein. The term “genetic engineering”refers to a recombinant DNA or RNA method used to create a host cellthat expresses a gene at elevated levels or at lowered levels, orexpresses a mutant form of the gene. In other words, the cell has beentransfected, transformed or transduced with a recombinant polynucleotidemolecule, and thereby altered so as to cause the cell to alterexpression of a desired polypeptide. For the purposes of the invention,the antibodies produced by a hybridoma cell line resulting from a cellfusion are not “recombinant polypeptides.” Further, viral polypeptidesproduced by a cell as a result of viral infection are also not“recombinant polypeptides” as meant herein unless the viral nucleic acidhas been altered by genetic engineering prior to infecting the cell. Themethods of “genetic engineering” also encompass numerous methodsincluding, but not limited to, amplifying nucleic acids using polymerasechain reaction, assembling recombinant DNA molecules by cloning them inEscherichia coli, restriction enzyme digestion of nucleic acids,ligation of nucleic acids, and transfer of bases to the ends of nucleicacids, among numerous other methods that are well-known in the art. Seee.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd)ed., vol. 1-3, Cold Spring Harbor Laboratory, 1989. Methods and vectorsfor genetically engineering cells and/or cell lines to express apolypeptide of interest are well known to those skilled in the art.Genetic engineering techniques include but are not limited to expressionvectors, targeted homologous recombination and gene activation (see, forexample, U.S. Pat. No. 5,272,071 to Chappel) and trans activation byengineered transcription factors (see e.g., Segal et al., 1999, Proc.Natl. Acad. Sci. USA 96(6):2758-63). Optionally, the polypeptides areexpressed under the control of a heterologous control element such as,for example, a promoter that does not in nature direct the production ofthat polypeptide. For example, the promoter can be a strong viralpromoter (e.g., CMV, SV40) that directs the expression of a mammalianpolypeptide. The host cell may or may not normally produce thepolypeptide. For example, the host cell can be a CHO cell that has beengenetically engineered to produce a human polypeptide, meaning thatnucleic acid encoding the human polypeptide has been introduced into theCHO cell. Alternatively, the host cell can be a human cell that has beengenetically engineered to produce increased levels of a humanpolypeptide normally present only at very low levels (e.g., by replacingthe endogenous promoter with a strong viral promoter).

“Growth phase” means a period during which cultured cells are rapidlydividing and increasing in number. During growth phase, cells aregenerally cultured in a medium and under conditions designed to maximizecell proliferation.

A “hybrid polar compound” is compound having two polar groups separatedby an apolar carbon chain. This includes hexamethylene bisacetamide(HMBA) and the other molecules discussed below and in the followingreferences: Richon et al. (1998), Proc. Natl. Acad. Sci. 95: 3003-07;Marks et al. (1994), Proc. Natl. Acad. Sci. 91: 10251-54; and U.S. Pat.Nos. 5,055,608 and 6,087,367.

The production of a polypeptide is “increased” by the addition of aninducing agent, such as hexamethylene bisacetamide (HMBA) or caffeine,if the amount the polypeptide produced in a culture containing theinducing agent is more than the amount of the polypeptide produced in anotherwise identical culture that does not contain the inducing agent.Similarly, the production of a polypeptide is “increased” by growth at atemperature other than 37° C. if the amount of polypeptide produced in aculture incubated at a temperature other than 37° C. is more than theamount of the polypeptide produced in an otherwise identical cultureincubated at 37° C.

A “multimerization domain” is a domain within a polypeptide moleculethat confers upon it a propensity to associate with other polypeptidemolecules through covalent or non-covalent interactions.

A “naturally-occurring polypeptide” is a polypeptide that occurs innature, that is, a polypeptide that can be produced by cells that havenot been genetically engineered. Such a polypeptide may also be producedby cells genetically engineered to produce it.

“Polypeptide” means a chain of at least 6 amino acids linked by peptidebonds. Optionally, a polypeptide can comprise at least 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids linked bypeptide bonds.

“Production medium” means a cell culture medium designed to be used toculture cells during a production phase.

“Production phase” means a period during which cells are producingmaximal amounts of recombinant polypeptide. A production phase ischaracterized by less cell division than during a growth phase and bythe use of medium and culture conditions designed to maximizepolypeptide production.

A “recombinant fusion polypeptide” is a fusion of all or part of atleast two polypeptides, which is made using the methods of geneticengineering.

A “recombinant polypeptide” is a polypeptide resulting from the processof genetic engineering. For the purposes of the invention, theantibodies produced by a hybridoma cell line resulting from a cellfusion are not “recombinant polypeptides.” Further, viral proteinsproduced by a cell as a result of viral infection with anaturally-occurring virus are also not “recombinant polypeptides” asmeant herein unless the viral nucleic acid has been altered by geneticengineering prior to infecting the cell.

“Substantially similar” polypeptides are at least 80%, optionally atleast 90%, identical to each other in amino acid sequence and maintainor alter in a desirable manner the biological activity of the unalteredpolypeptide. Conservative amino acid substitutions, unlikely to affectbiological activity, include, without limitation, the following: Ala forSer, Val for Ile, Asp for Glu, Thr for Ser, Ala for Gly, Ala for Thr,Ser for Asn, Ala for Val, Ser for Gly, Tyr for Phe, Ala for Pro, Lys forArg, Asp for Asn, Leu for Ile, Leu for Val, Ala for Glu, Asp for Gly,and these changes in the reverse. See e.g. Neurath et al., The Proteins,Academic Press, New York (1979). In addition exchanges of amino acidsamong members of the following six groups of amino acids will beconsidered to be conservative substitutions for the purposes of theinvention. The groups are: 1) methionine, alanine, valine, leucine, andisoleucine; 2) cysteine, serine, threonine, asparagine, and glutamine;3) aspartate and glutamate; 4) histidine, lysine, and arginine; 5)glycine and proline; and 6) tryptophan, tyrosine, and phenylalanine. Thepercent identity of two amino sequences can be determined by visualinspection and mathematical calculation, or more preferably, thecomparison is done by comparing sequence information using a computerprogram such as the Genetics Computer Group (GCG; Madison, Wis.)Wisconsin package version 10.0 program, ‘GAP’ (Devereux et al. (1984),Nucl. Acids Res. 12: 387) or other comparable computer programs. Thepreferred default parameters for the ‘GAP’ program includes: (1) theweighted amino acid comparison matrix of Gribskov and Burgess (1986),Nucl. Acids Res. 14: 6745, as described by Schwartz and Dayhoff, eds.,Atlas of polypeptide Sequence and Structure, National BiomedicalResearch Foundation, pp. 353-358 (1979), or other comparable comparisonmatrices; (2) a penalty of 30 for each gap and an additional penalty of1 for each symbol in each gap for amino acid sequences; (3) no penaltyfor end gaps; and (4) no maximum penalty for long gaps. Other programsused by those skilled in the art of sequence comparison can also beused.

“Transition phase” means a period of cell culture between a “growthphase” and a “production phase.” During transition phase, the medium andenvironmental conditions are typically shifted from those designed tomaximize proliferation to those designed to maximize polypeptideproduction.

A “variable antibody immunoglobulin domain” is an immunoglobulin domainthat is identical or substantially similar to a V_(L) or a V_(H) domainof human or animal origin.

The present invention is directed towards improved methods for culturingmammalian cells, which may have been genetically engineered to produce aparticular polypeptide. In particular, the invention is directed towardsculture methods that maximize the production of specific polypeptides.It is also directed towards methods of producing and obtaining suchpolypeptides from cultured mammalian cells. Polypeptides are useful in alarge variety of diagnostic, therapeutic, agricultural, nutritional, andresearch applications.

As shown by the experimental data reported herein, it has beendiscovered that xanthine derivatives and hybrid polar compounds usedseparately or together can dramatically induce the production ofrecombinant polypeptide from CHO cell lines. In particular, addition ofthe xanthine derivative caffeine to the production phase of a cellculture enhances recombinant polypeptide production. The hybrid polarcompound hexamethylene bisacetamide is also shown to be an effectiveinducer of recombinant polypeptide production. Further, other inducers,such as, for example, alkanoic acids, can also be added to either axanthine derivative, a hybrid polar compound, or both. Other methods,such as, for example, culturing the cells at temperatures from about 29°C. to about 36° C., between about 29° C. and 35° C., and/or from about30° C. to about 33° C. can also be used. Thus, the invention relates toinducing increased production of a recombinant polypeptide from a cellgrown in culture by exposing the cell to chemical inducers, includinghybrid polar compounds and/or xanthine derivatives.

The methods of the invention include culturing mammalian cells in mediumcomprising a hybrid polar compound, for example, hexamethylenebisacetamide (HMBA), optionally at temperatures between about 29° C. and35° C. or from about 30° C. to about 33° C. Other embodiments of theinvention encompass culture conditions in which an alkanoic acid and/ora xanthine, in addition to the hybrid polar compound, are added to theculture medium. In one embodiment, a xanthine and a hybrid polarcompound and culture temperatures between about 29° C. and 36° C. areused. Another embodiment comprises the addition of an alkanoic acid anda hybrid polar compound plus culture temperatures between about 29° C.and 36° C. Still another embodiment comprises addition of a xanthine, analkanoic acid, and a hybrid polar compound plus culture temperaturesbetween about 29° C. and 36° C. Optionally, cell culture using themethods of the invention can take place during a production phase, asdistinguished from a growth phase. A growth phase can be distinguishedfrom a production phase by, for example, a temperature shift and/or achange in medium such as, for example, the addition of one or moreinducers.

In one aspect, the invention provides a method comprising growing inculture a mammalian cell that has been genetically engineered to producea polypeptide; and adding to the culture a xanthine derivative. Agenetically engineered cell may be a cell that has been transformed witha recombinant vector encoding the polypeptide. In addition, thepolypeptide can be expressed under the control of a heterologouspromoter such as, for example, a CMV promoter. Typically, the cell doesnot naturally express the polypeptide or only naturally expresses thepolypeptide at very low levels (in the absence of genetic engineering).In another aspect, the invention provides a culture containing a cellgenetically engineered to produce a polypeptide, a production medium,and the xanthine derivative.

In addition, the methods and compositions of the invention can be usedin combination with any other known or yet to be discovered methods ofinducing the production of recombinant polypeptides. Such techniquesinclude cold temperature shift, alkanoic acid additions (as described inU.S. Pat. No. 5,705,364 to Etcheverry et al., incorporated herein byreference), DMF, and DMSO, to name just a few examples, as well as anyyet to be described and/or discovered induction techniques. As usedherein, “inducing” polypeptide production or “induction” refers toculturing cells under a set of conditions designed to maximize the totalamount of a desired polypeptide made by the cells. An “inducer” is anagent that, when added to culture medium, can increase the production ofa desired polypeptide in at least some cell lines. Combining theaddition of xanthine derivatives with other protein induction techniquescan have a synergistic effect on polypeptide induction, allowing forlower additions of xanthine derivatives and/or lower additions of otherinducing agents and/or more conservative temperature shifts. The othermethods of induction can take place at around the same time as xanthineaddition, and/or before and/or after xanthine addition. For example, onecan shift the temperature of the culture at day 0, and then add axanthine derivative and/or a hybrid polar compound, and optionally otherchemical inducers, later, e.g. one to several hours or days later. Sucha protocol allows some additional growth of a seeded culture before fullinduction. Furthermore, multiple additions of xanthine derivativesand/or hybrid polar compounds can be added to the culture during theproduction phase, separated by about 12, 24, 48, and/or 72 hours ormore, with or without additions of other inducing agents or changes inculture conditions. For example, an inducer can be added at day 0 andagain at day 4. Alternatively, an inducer can be added for the firsttime one, two, three, or four days after a temperature shift.

In one aspect, the invention entails performing a low temperature shift(shifting the temperature of the medium from the optimal growthtemperature, usually around 37° C., to a lower temperature, usually fromabout 29° C. to about 36° C., and optionally about 30° C. to about 34°C. at the time of, before, and/or after adding the xanthine derivativeor the hybrid polar compound. Alternatively, or in addition, an alkanoicacid or salt thereof (e.g. sodium butyrate) can be added to the cultureat around the same time as the xanthine derivative and/or hybrid polarcompound is added. Alkanoic acid can be added at concentrationstypically used for induction, or even at lower concentrations than wouldtypically be used. Thus, by manipulating both transcriptional andpost-transcriptional controls, higher levels of productivity may beachieved.

There are individual differences between cell lines in the effectivenessof various inducers. For example, although sodium butyrate is awidely-used inducer, it can have no effect or an adverse effect onpolypeptide production in some cell lines. See Table 5. Differentinducers or different concentrations of the same inducers may beappropriate for different cell lines. Furthermore, differenttemperatures may be appropriate for different cell lines. In spite ofthis variability, some inducers, such as, for example, caffeine,hexamethylene bisacetamide, and sodium butyrate, can be useful in a widevariety, though perhaps not all, cell lines.

Generally, xanthine derivatives have the structure illustrated below.

X, Y, and Z can be independently selected from a straight or branchedchain alkyl radical having from 1 to 12 carbons, a straight or branchedchain alkynyl radical having from 1 to 12 carbons (including a propynylradical), a straight or branched chain acyl radical having from 1 to 12carbons, a straight or branched chain radical with the structure —R-acylcontaining from 1 to 12 carbons where R is a saturated or unsaturatedaliphatic group, a straight or branched chain allenyl radical havingfrom 1 to 12 carbons, a straight or branched chain hydroxyalkyl radicalhaving from 1 to 12 carbons, a straight or branched chain hydroxyallenylradical having from 1 to 12 carbons, a straight or branched chainradical with the structure-allenyl-halogen having from 1 to 12 carbons,a cyclohexyl radical, and hydrogen. In some embodiments, at least one ofX, Y and Z is a methyl group. In some embodiments, each of X and Yindependently represents a hydrogen atom, a linear or branched alkylradical having up to 5 carbon atoms, an allyl radical, a propynylradical or a cyclohexyl radical, with the proviso that X and Y do notsimultaneously represent a hydrogen atom, and Z represents a hydrogen,methyl, ethyl, hydroxymethyl, hydroxyethyl or heterocyclo radical. Thesexanthines can be obtained using conventional processes and/or purchased.A number of different xanthine derivatives that can be used aredescribed in Beavo et al. (1970), Molec. Pharm. 6:597-603, andincorporated by reference herein.

Illustrative examples of xanthine derivatives that can be used in themethods and compositions of the invention include, but are not limitedto, caffeine (1,3,7-trimethylxanthine), theophylline(1,3-dimethylxanthine), theobromine (3,7-dimethylxanthine),3-isobutyl-1-methylxanthine, 3-butyl-1-methylxanthine,1,3,7-triethylxanthine, 3-cyclohexyl-1-ethylxanthine,3-ethyl-1-propynylxanthine, 3-ethyl-1-pentylxanthine, pentoxifylline,and aminophylline. Aminophylline is theophylline compound with1,2-ethylenediamine (2:1) dihydrate. Generally, the xanthine derivativeis added at a concentration in the culture from about 0.0005 to about 25millimolar, optionally from about 0.001 to about 10 millimolar, fromabout 0.005 to about 5 millimolar, or from about 0.01 to about 3millimolar. The optimal concentration of the xanthine derivative willvary depending upon its activity and the cell line, and can bedetermined by those skilled in the art using the guidance providedherein.

The xanthine derivative can be dissolved in any appropriate solvent. Forexample, 3-isobutyl-1-methylxanthine (IBX) can be dissolved in water,but must to be heated to almost the boiling point. Alternatively, IBXcan be dissolved in the solvents DMSO (dimethylsulfoxide), DMF(dimethylformamide), or DMA (dimethylacetamide). IBX can also be easilydissolved as a 100 millimolar stock solution in 0.5 M NaOH. Dilutions ofthis stock solution can be added to the induction media as it is beingprepared (pre-sterile) and the effects of the NaOH should beinconsequential since base must often be added to raise the pH of themedium to 7.0.

Many of the xanthine derivatives for use in the invention are cAMPphosphodiesterase inhibitors. Thus, in addition to using xanthinederivatives that are cAMP phosphodiesterase inhibitors, it is believedthat cAMP phosphodiesterase inhibitors that are not xanthine derivativescould also be used to induce polypeptide production in alternativemethods of the invention. Examples of such inducers include but are notlimited to imidazopyrimidine, pyrazolopyridine, etazolate,pyrazoloquinoline, and triazoloquinazoline (Pflugers Archiv 407: S31,1986). Other examples cAMP phosphodiesterase inhibitors can be found inU.S. Pat. No. RE 37,234, which is incorporated by reference herein.

The hybrid polar compounds, the use of which is encompassed by theinvention, can have two polar groups separated by a non-polar carbonchain, such as those described in Richon et al. (1998), Proc. Natl.Acad. Sci. 95: 3003-07, Marks et al. (1994), Proc. Natl. Acad. Sci. 91:10251-54, U.S. Pat. Nos. 5,055,608 and 6,087,367. The hybrid polarcompounds of the invention may have the property of inducing one or morechanges characteristic of a terminally differentiated state of the hostcells. These compounds include those with the structure:

R₁ and R₂ can be the same as or different from each other. R₁ and R₂ caneach be a carbonyl group to which a hydrogen atom, a hydroxyl group, asubstituted or unsubstituted, branched or unbranched alkyl, alkenyl,cycloalkyl, aryl, alkynl, allenyl, allyl, alkyloxy, aryloxy,arylalkyloxy, which contains 12 or fewer carbon atoms, or pyridinegroup, may also be attached. The “n” can be an integer from about fourto about eight. Specifically, HMBA is included within this class ofhybrid polar compounds, and its structure is:

The present invention further encompasses the use of hybrid polarcompounds with the following structure:

R₃ and R₄ can be the same as or different from each other. When R₃ andR₄ are the same, each is a substituted or unsubstituted arylamino,cycloalkylamino, pyridineamino, piperidino, 9-purine-6-amine, orthiozoleamino group containing 12 or fewer carbon atoms. Where R₃ and R₄are different, R₃ is equal to R₅—N—R₆, where R₅ and R₆ are the same asor different from each other and are a hydrogen atom, a hydroxyl group,a substituted or unsubstituted, branched or unbranched alkyl, alkenyl,cycloalkyl, aryl, alkynl, allenyl, allyl, alkyloxy, aryloxy,arylalkyloxy, or pyridine group, which contains 12 or fewer carbonatoms, or R₅ and R₆ bond together to form a piperidine group, and R₄ isa hydroxylamino, hydroxyl, amino, alkylamino, dialkylamino, or alkyloxygroup, which contains 12 or fewer carbon atoms. The “n” is an integerfrom about four to about eight.

The invention further embraces the use of all compounds disclosed inU.S. Pat. No. 6,087,367, U.S. Pat. No. 5,055,608, Richon et al., supra,and Marks et al., supra. In some of these, the apolar carbon chain maybe shorter than 4 carbons and longer than 8 carbons, and it may beinterrupted by aromatic groups, apolar groups, and/or polar groups.

If HMBA is used, it can be added at concentrations from about 0.1millimolar to about 20 millimolar, optionally, between about 0.1millimolar and about 5 millimolar. Other hybrid polar compounds may beactive at lower or higher concentrations. The optimal concentration fora particular hybrid polar compound will vary depending on its activityand the cell line in which it is used and can be determined by one ofskill in the art using routine methods and the guidance provided herein.For example, compounds such as suberoylanilide hydroxamic acid orm-carboxycinnamic acid bishydroxamide can be used at concentrationsabout one thousand fold lower than those required for HMBA, from about0.01 micromolar to about 10 micromolar. See Richon et al., supra.Concentrations of hybrid polar compounds required to induce celldifferentiation as disclosed in Marks et al. (supra) and Richon et al.(supra) can be used as a guide for determining the concentration of ahybrid polar compound required to enhance polypeptide production.Determination of the concentration needed for a specific hybrid polarcompound used in a specific cell line can be done using routine methodsas described herein and the guidance provided in Richon et al. (supra)and Marks et al. (supra).

The alkanoic acids for use in the invention include the selected acidand/or a corresponding salt. The acids include straight or branchedchain, saturated or unsaturated alkanoic acids or salts thereof. Analkanoic acid generally comprises from one to ten carbon atoms. Examplesof alkanoic acids contemplated by the invention are pentanoic acid,butyric acid, isobutyric acid, propionic acid, and acetic acid.Concentrations for alkanoic acids encompassed by the invention rangefrom about 0.05 millimolar to about 10 millimolar, optionally from about0.1 millimolar to about 2 millimolar. Appropriate concentrations ofalkanoic acids will vary depending upon their activity and the cell lineand can be determined by one of skill in the art using routine methodsand the guidance provided herein. An exemplary salt of butyric acid issodium butyrate. Appropriate salts of the alkanoic acids described aboveinclude those comprising sodium, potassium, or ammonium groups, amongothers.

Particularly preferred polypeptides for expression are polypeptide-baseddrugs, also known as biologics. Preferably, the polypeptides aresecreted as extracellular products. The polypeptide being produced cancomprise part or all of a polypeptide that is identical or substantiallysimilar to a naturally-occurring polypeptide, and/or it may, or may not,be a recombinant fusion polypeptide. Optionally, the polypeptide may bea human polypeptide, a fragment thereof, or a substantially similarpolypeptide that is at least 15 amino acids in length. It may comprise anon-antibody polypeptide and/or an antibody. It may be producedintracellularly or be secreted into the culture medium from which it canbe recovered. It may or may not be a soluble polypeptide.

The polypeptide being produced can comprise part or all of a polypeptidethat is identical or substantially similar to a naturally-occurringpolypeptide, and/or it may, or may not, be a recombinant fusionpolypeptide. It may comprise a non-antibody polypeptide and/or anantibody. It may be produced intracellularly or be secreted into theculture medium from which it can be recovered.

The invention can be used to induce the production of just about anypolypeptide, and is particularly advantageous for polypeptides whoseexpression is under the control of a strong promoter, such as forexample, a viral promoter, and/or polypeptides that are encoded on amessage that has an adenoviral tripartite leader element. Examples ofuseful expression vectors that can be used to produce proteins aredisclosed in International Application WO 01/27299 and in McMahan etal., (1991), EMBO J. 10: 2821, which describes the pDC409 vector. Aprotein is generally understood to be a polypeptide of at least about 10amino acids, optionally about 25, 75, or 100 amino acids.

Generally, the methods of the invention are useful for inducing theproduction of recombinant polypeptides. Some polypeptides that can beproduced with the methods of the invention include polypeptidescomprising amino acid sequences identical to or substantially similar toall or part of one of the following polypeptides: a flt3 ligand (asdescribed in International Application WO 94/28391, incorporarted hereinby reference), a CD40 ligand (as described in U.S. Pat. No. 6,087,329incorporated herein by reference), erythropoeitin, thrombopoeitin,calcitonin, leptin, IL-2, angiopoietin-2 (as described by Maisonpierreet al. (1997), Science 277(5322): 55-60, incorporated herein byreference), Fas ligand, ligand for receptor activator of NF-kappa B(RANKL, as described in International Application WO 01/36637,incorporated herein by reference), tumor necrosis factor (TNF)-relatedapoptosis-inducing ligand (TRAIL, as described in InternationalApplication WO 97/01633, incorporated herein by reference), thymicstroma-derived lymphopoietin, granulocyte colony stimulating factor,granulocyte-macrophage colony stimulating factor (GM-CSF, as describedin Australian Patent No. 588819, incorporated herein by reference), mastcell growth factor, stem cell growth factor (described in e.g. U.S. Pat.No. 6,204,363, incorporated herein by reference), epidermal growthfactor, keratinocyte growth factor, megakaryote growth and developmentfactor, RANTES, growth hormone, insulin, insulinotropin, insulin-likegrowth factors, parathyroid hormone, interferons including ainterferons, γ interferon, and consensus interferons (such as thosedescribed in U.S. Pat. Nos. 4,695,623 and 4,897,471, both of which areincorporated herein by reference), nerve growth factor, brain-derivedneurotrophic factor, synaptotagmin-like proteins (SLP 1-5),neurotrophin-3, glucagon, interleukins 1 through 18, colony stimulatingfactors, lymphotoxin-β, tumor necrosis factor (TNF), leukemia inhibitoryfactor, oncostatin-M, and various ligands for cell surface molecules ELKand Hek (such as the ligands for eph-related kinases or LERKS).Descriptions of polypeptides that can be produced according to theinventive methods may be found in, for example, Human Cytokines:Handbook for Basic and Clinical Research Vol. II (Aggarwal andGutterman, eds. Blackwell Sciences, Cambridge, Mass., 1998); GrowthFactors: A Practical Approach (McKay and Leigh, eds., Oxford UniversityPress Inc., New York, 1993); and The Cytokine Handbook (A. W. Thompson,ed., Academic Press, San Diego, Calif., 1991), all of which areincorporated herein by reference.

Other polypeptides that can be produced using the methods of theinvention include polypeptides comprising all or part of the amino acidsequence of a receptor for any of the above-mentioned polypeptides, anantagonist to such a receptor or any of the above-mentionedpolypeptides, and/or polypeptides substantially similar to suchreceptors or antagonists. These receptors and antagonists include: bothforms of tumor necrosis factor receptor (TNFR, referred to as p55 andp75, as described in U.S. Pat. No. 5,395,760 and U.S. Pat. No.5,610,279, both of which are incorporated herein by reference),Interleukin-1 (IL-1) receptors (types I and II; described in EP PatentNo. 0 460 846, U.S. Pat. No. 4,968,607, and U.S. Pat. No. 5,767,064, allof which are incorporated herein by reference), IL-1 receptorantagonists (such as those described in U.S. Pat. No. 6,337,072,incorporated herein by reference), IL-1 antagonists or inhibitors (suchas those described in U.S. Pat. Nos. 5,981,713, 6,096,728, and5,075,222, all of which are incorporated herein by reference) IL-2receptors, IL4 receptors (as described in EP Patent No. 0 367 566 andU.S. Pat. No. 5,856,296, both of which are incorporated by reference),IL-15 receptors, IL-17 receptors, IL-18 receptors,granulocyte-macrophage colony stimulating factor receptor, granulocytecolony stimulating factor receptor, receptors for oncostatin-M andleukemia inhibitory factor, receptor activator of NF-kappa B (RANK,described in WO 01/36637 and U.S. Pat. No. 6,271,349, both of which areincorporated by reference), osteoprotegerin (described in e.g. U.S. Pat.No. 6,015,938, incorporated by reference), receptors for TRAIL(including TRAIL receptors 1, 2, 3, and 4), and receptors that comprisedeath domains, such as Fas or Apoptosis-Inducing Receptor (AIR).

Other polypeptides that can be produced using the process of theinvention include polypeptides comprising all or part of the amino acidsequences of differentiation antigens (referred to as CD polypeptides)or their ligands or polypeptides substantially similar to either ofthese. Such antigens are disclosed in Leukocyte Typing VI (Proceedingsof the VIth International Workshop and Conference, Kishimoto, Kikutaniet al., eds., Kobe, Japan, 1996, which is incorporated by reference).Similar CD polypeptides are disclosed in subsequent workshops. Examplesof such antigens include CD22, CD27, CD30, CD39, CD40, and ligandsthereto (CD27 ligand, CD30 ligand, etc.). Several of the CD antigens aremembers of the TNF receptor family, which also includes 41BB and OX40.The ligands are often members of the TNF family, as are 41BB ligand andOX40 ligand. Accordingly, members of the TNF and TNFR families can alsobe purified using the present invention.

Enzymatically active polypeptides or their ligands can also be producedaccording to the methods of the invention. Examples include polypeptidescomprising all or part of one of the following polypeptides or theirligands or a polypeptide substantially similar to one of these:metalloproteinase-disintegrin family members, various kinases,glucocerebrosidase, superoxide dismutase, tissue plasminogen activator,Factor VIII, Factor IX, apolipoprotein E, apolipoprotein A-I, globins,an IL-2 antagonist, alpha-1 antitrypsin, TNF-alpha Converting Enzyme,ligands for any of the above-mentioned enzymes, and numerous otherenzymes and their ligands.

The methods of the invention can also be used to produce antibodies orportions thereof and chimeric antibodies, i.e. antibodies having humanconstant antibody immunoglobulin domains coupled to one or more murinevariable antibody immunoglobulin domain, fragments thereof, orsubstantially similar proteins. The method of the invention may also beused to produce conjugates comprising an antibody and a cytotoxic orluminescent substance. Such substances include: maytansine derivatives(such as DM1); enterotoxins (such as a Staphlyococcal enterotoxin);iodine isotopes (such as iodine-125); technium isotopes (such asTc-99m); cyanine fluorochromes (such as Cy5.5.18); andribosome-inactivating polypeptides (such as bouganin, gelonin, orsaporin-S6). The invention can also be used to produce chimeric proteinsselected in vitro to bind to a specific target protein and modify itsactivity such as those described in International Applications WO01/83525 and WO 00/24782, both of which are incorporated by reference.Examples of antibodies, in vitro-selected chimeric proteins, orantibody/cytotoxin or antibody/luminophore conjugates that can beproduced by the methods of the invention include those that recognizeany one or a combination of polypeptides including, but not limited to,the above-mentioned proteins and/or the following antigens: CD2, CD3,CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44,CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1a, IL-1 p, IL-2, IL-3, IL-7,IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL4 receptor, IL-6 receptor,IL-13 receptor, IL-18 receptor subunits, PDGF-β and analogs thereof(such as those described in U.S. Pat. Nos. 5,272,064 and 5,149,792),VEGF, TGF, TGF-β2, TGF-β1, EGF receptor (including those described inU.S. Pat. No. 6,235,883 B1, incorporated by reference) VEGF receptor,hepatocyte growth factor, osteoprotegerin ligand, interferon gamma, Blymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1, andzTNF4; see Do and Chen-Kiang (2002), Cytokine Growth Factor Rev. 13(1):19-25), C5 complement, IgE, tumor antigen CA1125, tumor antigen MUC 1,PEM antigen, LCG (which is a gene product that is expressed inassociation with lung cancer), HER-2, a tumor-associated glycoproteinTAG-72, the SK-1 antigen, tumor-associated epitopes that are present inelevated levels in the sera of patients with colon and/or pancreaticcancer, cancer-associated epitopes or polypeptides expressed on breast,colon, squamous cell, prostate, pancreatic, lung, and/or kidney cancercells and/or on melanoma, glioma, or neuroblastoma cells, the necroticcore of a tumor, integrin alpha 4 beta 7, the integrin VLA4, B2integrins, TRAIL receptors 1, 2, 3, and 4, RANK, RANK ligand, TNF-α, theadhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM),intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesin, theplatelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain,parathyroid hormone, rNAPc2 (which is an inhibitor of factor VIIa-tissuefactor), MHC I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP),tumor necrosis factor (TNF), CTLA4 (which is a cytotoxic Tlymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10 beta, HLA-DRantigen, L-selectin, Respiratory Syncitial Virus, human immunodeficiencyvirus (HIV), hepatitis B virus (HBV), Streptococcus mutans, andStaphlycoccus aureus.

The invention may also be used to produce all or part of ananti-idiotypic antibody or a substantially similar polypeptide,including anti-idiotypic antibodies against: an antibody targeted to thetumor antigen gp72; an antibody against the ganglioside GD3; an antibodyagainst the ganglioside GD2; or antibodies substantially similar tothese.

The methods of the invention can also be used to produce recombinantfusion polypeptides comprising any of the above-mentioned polypeptides.For example, recombinant fusion polypeptides comprising one of theabove-mentioned polypeptides plus a multimerization domain, such as aleucine zipper, a coiled coil, an Fc portion of an antibody, or asubstantially similar protein, can be produced using the methods of theinvention. See e.g. WO94/10308; Lovejoy et al. (1993), Science259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury etal. (1994), Nature 371:80-83; Hakansson et al. (1999), Structure7:255-64, all of which are incorporated by reference. Specificallyincluded among such recombinant fusion polypeptides are polypeptides inwhich a portion of TNFR or RANK is fused to an Fc portion of an antibody(TNFR:Fc or RANK:Fc). TNFR:Fc comprises the Fc portion of an antibodyfused to an extracellular domain of TNFR, which includes amino acidsequences substantially similar to amino acids 1-163, 1-185, or 1-235 ofFIG. 2A of U.S. Pat. No. 5,395,760, which is incorporated by reference.RANK:Fc is described in International Application WO 01/36637, which isincorporated by reference.

Preferably, the polypeptides are expressed under the control of aheterologous control element such as, for example, a promoter that doesnot in nature direct the production of that polypeptide. For example,the promoter can be a strong viral promoter (e.g., CMV, SV40) thatdirects the expression of a mammalian polypeptide. The host cell may ormay not normally produce the polypeptide. For example, the host cell canbe a CHO cell that has been genetically engineered to produce a humanpolypeptide, meaning that nucleic acid encoding the human polypeptidehas been introduced into the CHO cell. Alternatively, the host cell canbe a human cell that has been genetically engineered to produceincreased levels of a human polypeptide normally present only at verylow levels (e.g., by replacing the endogenous promoter with a strongviral promoter). For the production of recombinant polypeptides, anexpression vector encoding the recombinant polypeptide can betransferred, for example by transfection or viral infection, into asubstantially homogeneous culture of host cells. The expression vector,which can be constructed using the methods of genetic engineering, caninclude nucleic acids encoding the polypeptide of interest operablylinked to suitable regulatory sequences.

The regulatory sequences are typically derived from mammalian,microbial, viral, and/or insect genes. Examples of regulatory sequencesinclude transcriptional promoters, operators, and enhancers, a ribosomalbinding site (see e.g. Kozak (1991), J. Biol. Chem. 266:19867-19870),appropriate sequences to control transcriptional and translationalinitiation and termination, polyadenylation signals (see e.g. McLauchlanet al. (1988), Nucleic Acids Res. 16:5323-33), and matrix and scaffoldattachment sites (see Phi-Van et al. (1988), Mol. Cell. Biol.10:2302-07; Stief et al. (1989), Nature 341:342-35; Bonifer et al.(1990), EMBO J. 9:2843-38). Nucleotide sequences are operably linkedwhen the regulatory sequence functionally relates to the polypeptidecoding sequence. Thus, a promoter nucleotide sequence is operably linkedto a polypeptide coding sequence if the promoter nucleotide sequencecontrols the transcription of the coding sequence. A gene encoding aselectable marker is generally incorporated into the expression vectorto facilitate the identification of recombinant cells.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter and enhancer sequences are derived from polyoma virus,adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV).For example, the human CMV promoter/enhancer of immediate early gene 1may be used. See e.g. Patterson et al. (1994), Applied Microbiol.Biotechnol. 40:691-98. DNA sequences derived from the SV40 viral genome,for example, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites can be used to provide other genetic elements forexpression of a structural gene sequence in a mammalian host cell. Viralearly and late promoters are particularly useful because both are easilyobtained from a viral genome as a fragment, which can also contain aviral origin of replication (Fiers et al. (1978), Nature 273:113;Kaufman (1990), Meth. in Enzymol. 185:487-511). Smaller or larger SV40fragments can also be used, provided the approximately 250 bp sequenceextending from the Hind III site toward the Bgl I site located in theSV40 viral origin of replication site is included.

In addition, a sequence encoding an appropriate native or heterologoussignal peptide (leader sequence) can be incorporated into the expressionvector, to promote extracellular secretion of the recombinantpolypeptide. The signal peptide will be cleaved from the recombinantpolypeptide upon secretion from the cell. The choice of signal peptideor leader depends on the type of host cells in which the recombinantpolypeptide is to be produced. Examples of signal peptides that arefunctional in mammalian host cells include the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195, the signalsequence for interleukin-2 receptor described in Cosman et al. (1984),Nature 312:768; the interleukin-4 receptor signal peptide described inEP Patent No. 367,566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; and the type II interleukin-1receptor signal peptide described in EP Patent No. 0 460 846.

Established methods for introducing DNA into mammalian cells have beendescribed. Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15-69. Additional protocols using commercially available reagents, suchas the cationic lipid reagents LIPOFECTAMINE™, LIPOFECTAMINE™ 2000, orLIPOFECTAMINE™-PLUS (which can be purchased from Invitrogen), can beused to transfect cells. Felgner et al. (1987)., Proc. Natl. Acad. Sci.USA 84:7413-7417. In addition, electroporation or bombardment withmicroprojectiles coated with nucleic acids can be used to transfectmammalian cells using procedures, such as those in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed. Vol. 1-3, Cold SpringHarbor Laboratory Press (1989) and Fitzpatrick-McElligott (1992),Biotechnology (NY) 10(9):103640. Selection of stable transformants canbe performed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al. ((1990), Meth. inEnzymology 185:487-511), describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-BII, which is deficient in DHFR.Urlaub and Chasin (1980), Proc. Natl. Acad. Sci. USA 77:42164220. Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., in Animal Cell Technology, pp. 529-534 (1997); U.S. Pat.Nos. 6,312,951 B1, 6,027,915, and 6,309,841 B1) and the tripartiteleader (TPL) and VA gene RNAs from Adenovirus 2 (Gingeras et al. (1982),J. Biol. Chem. 257:13475-13491). The internal ribosome entry site (IRES)sequences of viral origin allows dicistronic mRNAs to be translatedefficiently (Oh and Sarnow (1993), Current Opinion in Genetics andDevelopment 3:295-300; Ramesh et al. (1996), Nucleic Acids Research24:2697-2700). Expression of a heterologous cDNA as part of adicistronic mRNA followed by the gene for a selectable marker (e.g.DHFR) has been shown to improve transfectability of the host andexpression of the heterologous cDNA (Kaufman et al. (1990), Methods inEnzymol. 185:487-511). Exemplary expression vectors that employdicistronic mRNAs are pTR-DC/GFP described by Mosser et al.,Biotechniques 22:150-161 (1997), and p2A5I described by Morris et al.,in Animal Cell Technology, pp. 529-534 (1997).

A useful high expression vector, pCAVNOT, has been described by Mosleyet al. ((1989), Cell 59:335-348). Other expression vectors for use inmammalian host cells can be constructed as disclosed by Okayama and Berg((1983), Mol. Cell. Biol. 3:280). A useful system for stable high levelexpression of mammalian cDNAs in C127 murine mammary epithelial cellscan be constructed substantially as described by Cosman et al. ((1986),Mol. Immunol. 23:935). A useful high expression vector, PMLSV N1/N4,described by Cosman et al. ((1984), Nature 312:768), has been depositedas ATCC 39890. Additional useful mammalian expression vectors aredescribed in EP Patent No.-A-0 367 566 and WO 01/27299 A1. The vectorscan be derived from retroviruses. In place of the native signalsequence, a heterologous signal sequence can be added, such as one ofthe following sequences: the signal sequence for IL-7 described in U.S.Pat. No. 4,965,195; the signal sequence for IL-2 receptor described inCosman et al. (Nature 312:768 (1984)); the IL4 signal peptide describedin EP Patent No. 0 367 566; the type I IL-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; and the type II IL-1 receptorsignal peptide described in EP Patent No. 0 460 846.

The polypeptides can be produced recombinantly in eukaryotic cells andare preferably secreted by host cells adapted to grow in cell culture.Optionally, host cells for use in the invention are preferably mammaliancells. The cells can be also genetically engineered to express a gene ofinterest, can be mammalian production cells adapted to grow in cellculture, and/or can be homogenous cell lines. Examples of such cellscommonly used in the industry are VERO, BHK, HeLa, CV1 (including Cos),MDCK, 293, 3T3, myeloma cell lines (e.g., NSO, NS1), PC12, W138 cells,and Chinese hamster ovary (CHO) cells, which are widely used for theproduction of several complex recombinant polypeptides, e.g. cytokines,clotting factors, and antibodies (Brasel et al. (1996), Blood88:2004-2012; Kaufman et al. (1988), J. Biol Chem 263:6352-6362;McKinnon et al. (1991), J Mol Endocrinol 6:231-239; Wood et al. (1990),J. Immunol. 145:3011-3016). The dihydrofolate reductase (DHFR)-deficientmutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220, which is incorporated by reference), DXB11 and DG44, aredesirable CHO host cell lines because the efficient DHFR selectable andamplifiable gene expression system allows high level recombinantpolypeptide expression in these cells (Kaufman R. J. (1990), MethEnzymol 185:537-566, which is incorporated by reference). In addition,these cells are easy to manipulate as adherent or suspension culturesand exhibit relatively good genetic stability. CHO cells and recombinantpolypeptides expressed in them have been extensively characterized andhave been approved for use in clinical commercial manufacturing byregulatory agencies. The methods of the invention can also be practicedusing hybridoma cell lines that produce an antibody. Methods for makinghybridoma lines are well known in the art. See e.g. Berzofsky et al. inPaul, ed., Fundamental Immunology, Second Edition, pp. 315-356, at347-350, Raven Press Ltd., New York (1989). Cell lines derived from theabove-mentioned lines are also suitable for practicing the invention.

According to the present invention, a mammalian host cell is culturedunder conditions that promote the production of the polypeptide ofinterest, which can be an antibody or a recombinant polypeptide. Basalcell culture medium formulations are well known in the art. To thesebasal culture medium formulations the skilled artisan will addcomponents such as amino acids, salts, sugars, vitamins, hormones,growth factors, buffers, antibiotics, lipids, trace elements and thelike, depending on the requirements of the host cells to be cultured.The culture medium may or may not contain serum and/or protein. Varioustissue culture media, including serum-free and/or defined culture media,are commercially available for cell culture. Tissue culture media isdefined, for purposes of the invention, as a media suitable for growthof animal cells, and preferably mammalian cells, in in vitro cellculture. Typically, tissue culture media contains a buffer, salts,energy source, amino acids, vitamins and trace essential elements. Anymedia capable of supporting growth of the appropriate eukaryotic cell inculture can be used; the invention is broadly applicable to eukaryoticcells in culture, particularly mammalian cells, and the choice of mediais not crucial to the invention. Tissue culture media suitable for usein the invention are commercially available from, e.g., ATCC (Manassas,Va.). For example, any one or combination of the following media can beused: RPMI-1640 Medium, RPMI-1641 Medium, Dulbecco's Modified Eagle'sMedium (DMEM), Minimum Essential Medium Eagle, F-12K Medium, Ham's F12Medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A Medium,Leibovitz's L-15 Medium, and serum-free media such as EX-CELL™ 300Series (available from JRH Biosciences, Lenexa, Kans., USA), amongothers, which can be obtained from the American Type Culture Collectionor JRH Biosciences, as well as other vendors. When defined medium thatis serum-free and/or peptone-free is used, the medium is usually highlyenriched for amino acids and trace elements. See, for example, U.S. Pat.No. 5,122,469 to Mather et al. and U.S. Pat. No. 5,633,162 to Keen etal.

In the methods and compositions of the invention, cells can be grown inserum-free, protein-free, growth factor-free, and/or peptone-free media.The term “serum-free” as applied to media includes any mammalian cellculture medium that does not contain serum, such as fetal bovine serum.The term “insulin-free” as applied to media includes any medium to whichno exogenous insulin has been added. By exogenous is meant, in thiscontext, other than that produced by the culturing of the cellsthemselves. The term “IGF-1-free” as applied to media includes anymedium to which no exogenous Insulin-like growth factor-1 (IGF-1) oranalog (such as, for example, LongR3, [Ala31], or [Leu24][Ala31] IGF-1analogs available from GroPep Ltd. of Thebarton, South Australia) hasbeen added. The term “growth-factor free” as applied to media includesany medium to which no exogenous growth factor (e.g., insulin, IGF-1)has been added. The term “protein-free” as applied to media includesmedium free from exogenously added protein, such as, for example,transferrin and the protein growth factors IGF-1 and insulin.Protein-free media may or may not have peptones. The term “peptone-free”as applied to media includes any medium to which no exogenous proteinhydrolysates have been added such as, for example, animal and/or plantprotein hydrolysates. Eliminating peptone from media has the advantagesof reducing lot to lot variability and enhancing processing such asfiltration. Chemically defined media are media in which every componentis defined and obtained from a pure source, preferably a non-animalsource.

The skilled artisan may also choose to use one of the manyindividualized media formulations that have been developed to maximizecell growth, cell viability, and/or recombinant polypeptide productionin a particular cultured host cell. The methods according to the currentinvention may be used in combination with commercially available cellculture media or with a cell culture medium that has been individuallyformulated for use with a particular cell line. For example, an enrichedmedium that could support increased polypeptide production may comprisea mixture of two or more commercial media, such as, for instance, DMEMand Ham's F1 2 media combined in ratios such as, for example, 1:1, 1:2,1:3, 1:4, 1:5, 1:6, 1:7, 1:8, or even up to 1:15 or higher.Alternatively or in addition, a medium can be enriched by the additionof nutrients, such as amino acids or peptone, and/or a medium (or mostof its components with the exceptions noted below) can be used atgreater than its usual, recommended concentration, for example at 2×,3×, 4×, 5×, 6×, 7×, 8×, or even higher concentrations. As used herein,“1×” means the standard concentration, “2X” means twice the standardconcentration, etc. In any of these embodiments, medium components thatcan substantially affect osmolarity, such as salts, cannot be increasedin concentration so that the osmolarity of the medium falls outside ofan acceptable range. Thus, a medium may, for example, be 8× with respectto all components except salts, which can be present at only 1×. Anenriched medium may be serum free and/or protein free. Further, a mediummay be supplemented periodically during the time a culture is maintainedto replenish medium components that can become depleted such as, forexample, vitamins, amino acids, and metabolic precursors. As is known inthe art, different media and temperatures may have somewhat differenteffects on different cell lines, and the same medium and temperature maynot be suitable for all cell lines.

Suitable culture conditions for mammalian cells are known in the art.See e.g. Animal cell culture: A Practical Approach, D. Rickwood, ed.,Oxford university press, New York (1992). Mammalian cells may becultured in suspension or while attached to a solid substrate.Furthermore, mammalian cells may be cultured, for example, in fluidizedbed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks,or stirred tank bioreactors, with or without microcarriers, and operatedin a batch, fed batch, continuous, semi-continuous, or perfusion mode.

The methods according to the present invention may be used to improvethe production of recombinant polypeptides in both single phase andmultiple phase culture processes. In a single phase process, cells areinoculated into a culture environment and the disclosed methods areemployed during the single production phase. In a multiple stageprocess, cells are cultured in two or more distinct phases. For examplecells may be cultured first in a growth phase, under environmentalconditions that maximize cell proliferation and viability, thentransferred to a production phase, under conditions that maximizepolypeptide production. The growth and production phases may be precededby, or separated by, one or more transition phases. In multiple phaseprocesses the methods according to the present invention are employed atleast during the production phase. A growth phase may occur at a highertemperature than a production phase. For example, a growth phase mayoccur at a first temperature from about 35° C. to about 38° C., and aproduction phase may occur at a second temperature from about 29° C. toabout 36° C., optionally from about 30° C. to about 33° C. Chemicalinducers of polypeptide production, such as, for example, caffeine,butyrate, and HMBA, may be added at the same time as, before, and/orafter a temperature shift. If inducers are added after a temperatureshift, they can be added from one hour to five days after thetemperature shift, optionally from one to two days after the temperatureshift.

After induction using the methods of the invention, the resultingexpressed polypeptide can then be collected. In addition, thepolypeptide can purified, or partially purified, from such culture orcomponent (e.g., from culture medium or cell extracts or bodily fluid)using known processes. By “partially purified” means that somefractionation procedure, or procedures, have been carried out, but thatmore polypeptide species (at least 10%) than the desired polypeptide ispresent. By “purified” is meant that the polypeptide is essentiallyhomogeneous, i.e., less than 1% contaminating polypeptides are present.Fractionation procedures can include but are not limited to one or moresteps of filtration, centrifugation, precipitation, phase separation,affinity purification, gel filtration, ion exchange chromatography,hydrophobic interaction chromatography (HIC; using such resins as phenylether, butyl ether, or propyl ether), HPLC, or some combination ofabove.

For example, the purification of the polypeptide can include an affinitycolumn containing agents which will bind to the polypeptide; one or morecolumn steps over such affinity resins as concanavalin A-agarose,heparin-TOYOPEARL® (Toyo Soda Manufacturing Co., Ltd., Japan) orCibacrom blue 3GA SEPHAROSE® (Pharmacia Fine Chemicals, Inc., New York);one or more steps involving elution; and/or immunoaffinitychromatography. The polypeptide can be expressed in a form thatfacilitates purification. For example, it may be expressed as a fusionpolypeptide, such as those of maltose binding polypeptide (MBP),glutathione-S-transferase (GST), or thioredoxin (TRX). Kits forexpression and purification of such fusion polypeptides are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and InVitrogen, respectively. The polypeptide can betagged with an epitope and subsequently purified by using a specificantibody directed to such epitope. One such epitope (FLAG®) iscommercially available from Kodak (New Haven, Conn.). It is alsopossible to utilize an affinity column comprising a polypeptide-bindingprotein, such as a monoclonal antibody to the recombinant polypeptide,to affinity-purify expressed polypeptides. Other types of affinitypurification steps can be a Protein A or a Protein G column, whichaffinity agents bind to proteins that contain Fc domains. Polypeptidescan be removed from an affinity column using conventional techniques,e.g. in a high salt elution buffer and then dialyzed into a lower saltbuffer for use or by changing pH or other components depending on theaffinity matrix utilized, or can be competitively removed using thenaturally occurring substrate of the affinity moiety.

The desired degree of final purity depends on the intended use of thepolypeptide. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no polypeptide bandscorresponding to other polypeptides are detectable upon analysis bySDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognizedby one skilled in the pertinent field that multiple bands correspondingto the polypeptide can be visualized by SDS-PAGE, due to differentialglycosylation, differential post-translational processing, and the like.Optionally, the polypeptide of the invention is purified to substantialhomogeneity, as indicated by a single polypeptide band upon analysis bySDS-PAGE. The polypeptide band can be visualized by silver staining,Coomassie blue staining, or (if the polypeptide is radiolabeled) byautoradiography.

The invention also optionally encompasses further formulating thepolypeptides. By the term “formulating” is meant that the polypeptidescan be buffer exchanged, sterilized, bulk-packaged, and/or packaged fora final user. For purposes of the invention, the term “sterile bulkform” means that a formulation is free, or essentially free, ofmicrobial contamination (to such an extent as is acceptable for foodand/or drug purposes), and is of defined composition and concentration.The term “sterile unit dose form” means a form that is appropriate forthe customer and/or patient administration or consumption. Suchcompositions can comprise an effective amount of the polypeptide, incombination with other components such as a physiologically acceptablediluent, carrier, or excipient. The term “physiologically acceptable”means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredient(s).

Formulations suitable for administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the recipient; and aqueous and non-aqueous sterilesuspensions which may include suspending agents or thickening agents.The polypeptides can be formulated according to known methods used toprepare pharmaceutically useful compositions. They can be combined inadmixture, either as the sole active material or with other known activematerials suitable for a given indication, with pharmaceuticallyacceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphatebuffered solutions), preservatives (e.g., thimerosal, benzyl alcohol,parabens), emulsifiers, solubilizers, adjuvants, and/or carriers.Suitable formulations for pharmaceutical compositions include thosedescribed in Remington's Pharmaceutical Sciences, 16th ed. 1980, MackPublishing Company, Easton, Pa. In addition, such compositions can becomplexed with polyethylene glycol (PEG), metal ions, or incorporatedinto polymeric compounds such as polyacetic acid, polyglycolic acid,hydrogels, dextran, etc., or incorporated into liposomes,microemulsions, micelles, unilamellar or multilamellar vesicles,erythrocyte ghosts or spheroblasts. Suitable lipids for liposomalformulation include, without limitation, monoglycerides, diglycerides,sulfatides, lysolecithin, phospholipids, saponin, bile acids, and thelike. Preparation of such liposomal formulations is within the level ofskill in the art, as disclosed, for example, in U.S. Pat. Nos.4,235,871,4,501,728, 4,837,028, and 4,737,323. Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance, and are thus chosen according tothe intended application, so that the characteristics of the carrierwill depend on the selected route of administration. Sustained-releaseforms suitable for use include, but are not limited to, polypeptidesthat are encapsulated in a slowly-dissolving biocompatible polymer (suchas the alginate microparticles described in U.S. Pat. No. 6,036,978),admixed with such a polymer (including topically applied hydrogels), andor encased in a biocompatible semi-permeable implant.

The invention having been described, the following examples are offeredby way of illustration, and not limitation.

EXAMPLE 1 Comparison of the Inducing Activity of Caffeine and Butyrateat 31° C.

In this experiment, caffeine (at concentrations from 0.5 to 2.0 mM) wascompared to sodium butyrate for its ability to induce expression of arecombinant polypeptide. A CHO cell production line geneticallyengineered to express TNFR:Fc (cell line #5) was used to test theeffectiveness of caffeine as an inducing agent. CHO cells were grown inspinner flasks at 37° C. using serum-free growth medium containingmethotrexate. When the appropriate cell mass was obtained, the cellswere placed into induction conditions by a five minute centrifugation at1000×g, followed by replacement of the growth medium with serum-freemedium without methotrexate. The cells, at initial cell densities of2×10⁶ cells/ml in 20 ml, were placed in 125 ml plastic Erlenmeyer flaskswith plug seal caps and placed on shaker platforms in incubators set tothe appropriate temperatures. Cell viability and number were monitoredby haemocytometer counting using trypan blue dye. Recombinantpolypeptide titers were assessed by ELISA-based assays.

For this cell line, 0.2 mM was known to be the optimal concentration ofsodium butyrate for induction. Accordingly, 0.2 mM sodium butyrate wascompared against the inducing effects of 0.5, 1.0, and 2.0 mM caffeine.A flask containing no inducing compound was also included. The shakerflasks were incubated in this induction phase for 5 days at 31° C. inincubators without carbon dioxide control.

After 5 days in culture, cell viabilities for all of the testedconditions were very similar and ranged between 75 and 85%. The highestrelative protein titer (in μg/ml), which was about 1.15 times the titerof the control culture without inducers, and relative productivity (inμg protein/10⁶ cells/day), which was about 1.3 times the productivity ofthe control culture, was exhibited by the cells that were induced with 1mM caffeine. Cells induced with 0.2 mM butyrate produced about 1.11times the total protein (in μg/ml) produced by the control culture at arate (in μg protein/10⁶ cells/day) that was about 1.07 times the rate ofcontrol cultures. Similar protein titers were observed in the cellsinduced with 0.5 mM caffeine, although these cultures had slightlyhigher rates of production. At caffeine concentrations of 2 mM, proteintiter was similar to that observed with no inducing agent, although therate of productivity per cell was higher.

These results indicate that caffeine can be used as an inducing agentand can induce product titers equal or exceeding those observed usingsodium butyrate as an inducing agent. In addition, further experimentaldata was obtained which indicated that recombinant polypeptide producedusing caffeine was equal in product quality (e.g., glycosylation,folding, and amino acid composition) to that produced using sodiumbutyrate.

EXAMPLE 2 Induction of Recombinant Polypeptide Expression in Cell Line#9

In this experiment, the effect of caffeine (at concentrations from 0 to1.4 mM) on the induction of expression of a different recombinantpolypeptide, a soluble form of the IL-1 receptor type II, in a secondCHO cell line (cell line #9) was examined.

CHO cells were grown in spinner flasks at 37° C. using serum-free growthmedium containing methotrexate. When the appropriate cell mass wasobtained, spent medium was removed by a five minute centrifugation at1000×g and replaced with production medium without methotrexate. Thecells, with initial cell densities of 2×10⁶ cells/ml in 20 ml, wereplaced in 125 ml plastic Erlenmeyer flasks with plug seal caps. Thefollowing caffeine concentrations were tested: 0, 0.6, 0.8, 1.0, 1.2,and 1.4 mM caffeine. The flasks were then incubated in this inductionphase for 5 days at 31° C. in incubators without carbon dioxide control.Cell viability and number were monitored by haemocytometer countingusing trypan blue dye. Recombinant polypeptide titers were assessed byELISA-based assays. Each induction assessment experiment was carried outfor 5 days.

After 5 days in culture, cell viability for most of the testedconditions was similar and averaged around 85%. FIG. 1. For the flaskinduced with 1.4 mM caffeine, 67% cell viability was observed after 5days. Similar protein titers were observed using 0.6 mM, 0.8 mM, and 1.0mM caffeine, that is, about 350 μg/mL, which is equal to the titerobserved for 0.5 mM butyrate. FIG. 2. Since 0.6 mM is the lowestcaffeine concentration tested, these data do not exclude the possibilitythat even lower concentrations of caffeine might give equal or betterresults. The highest productivity (in μg protein/10⁶ cells/day) observedfor a caffeine-induced culture was in the 0.8 mM caffeine culture. FIG.3. At higher levels of caffeine, i.e., 1.2 and 1.4 mM, protein titerswere comparable to the negative control (no inducing agent), althoughproductivity on a per cell basis was somewhat higher. FIGS. 2 and 3.

EXAMPLE 3 Induction Of Recombinant Polypeptide Expression in Cell Line#60

In this experiment, the use of caffeine to induce recombinant productionfrom a third CHO cell line (cell line #60) expressing a thirdrecombinant product, a human antibody that recognizes epidermal growthfactor receptor, was analyzed. For this cell line, the inducing effectsof 0, 0.5, 1.0, 1.5, and 2.0 mM caffeine were tested, and the experimentwas conducted as in the previous experiment except that the inductionphase was performed at 36° C.

At day 5, the flask of cells with no inducer and the flask of cellsinduced with 0.5 mM caffeine exhibited the highest cell viabilities(about 76%) of all the conditions. Viabilities of cultures containing1.0 mM and 1.5 mM caffeine were about 68% and 60%, respectively.Cultures containing 0.75 mM butyrate or 2.0 mM caffeine were about 51%viable. Thus viability, overall, was lower than that seen in cell line#9 at 5 days, an effect that might be attributed to a variety of factorsincluding the difference in temperature and/or cell line differences. Aclear dose-response was observed with higher caffeine concentrationsleading to lower cell viabilities.

The highest day 5 protein titer was observed in cells induced by 0.5 mMcaffeine (305 μg/ml), which was about 111% of the titer of the controlculture with no inducer. Generally, the titer of recombinant polypeptidewas less as caffeine concentrations increased above 0.5 mM. Productivity(in μg protein/10⁶ cells/day) appeared to be linked to caffeineconcentration, with the highest productivity obtained from cells inducedwith 2.0 mM caffeine and a lower level of productivity obtained from thecells induced with lower caffeine concentrations. Since a 0.5 mM was thelowest caffeine concentration tested as well as the most effectiveconcentration tested for the induction of protein production, these datado not exclude the possibility that a lower concentration of caffeinemight be equally or more effective as an inducer of cell line #60incubated at 36° C.

This experiment, along with those described in Examples 1 and 2,demonstrates that the ability of caffeine to induce recombinantpolypeptide expression is not cell line-specific and that favorable cellviability is maintained in caffeine's presence. In addition, caffeinecan be used in an induction or production phase implemented attemperatures from 31° C. to 36° C. However, these data also indicatedifferences between cell lines in how effectively caffeine induces thesynthesis of a recombinant protein. For example, induction of cell line#9 with caffeine is more effective than induction of cell line #60.Compare Example 2 and FIG. 2 to Example 3.

EXAMPLE 4 Optimization of Induction for Cell Line #60

The purpose of this experiment was to test ranges of temperature andcaffeine concentrations in shake flasks in order to optimize theinduction conditions for the cell line #60.

Materials and Methods. Twelve shaker flasks were set up under theconditions described in Table 1. TABLE 1 Caffeine Concentrations andTemperatures of Samples Flask Temperature Caffeine Number (° C.) (mM) 136 0 2 36 0.5 3 36 1.0 4 36 1.5 5 36 2.0 6 36 2.5 7 37 0 8 37 0.5 9 371.0 10 37 1.5 11 37 2.0 12 37 2.5Cells were collected via centrifugation from a spinner culture of cellline #60 (26.85×10⁵ cells/ml, 95.2% viable) and inoculated into a 575 mlspinner flask at 2×10⁶ cells/ml in serum-free production medium. Theculture was then aliquoted into twelve shake flasks. Caffeine was addedaccording to the experimental plan described in Table 1. The shakeflasks were incubated at the designated temperatures for 7 days. Sampleswere taken on days 3, 5 and 7. Cell density and viability were measuredusing an automated system of cell counting that employs trypan bluestaining to determine viability (the Cell Density Examination System orCedex, developed by innovatis GmbH, Bielefeld, Germany). Glucose andlactate measurements were taken with the Yellow Springs Instruments 2700Select (available from Yellow Springs Instruments, Yellow Springs, Ohio,USA). Glucose was added on demand to maintain a concentration of >2 g/l.CO₂ and external pH were measured using the Ciba-Corning 248 blood gasanalyzer (available from Bayer Diagnostics, Tarryton, N.Y., USA).Protein titers were determined via a pre-purification of the antibody ona Protein A column followed by a measurement of the absorbance of theprotein bound and eluted from the column at 280 nanometers. Cumulativeviable cell densities (CVCDs) were calculated as follows: the CVCD forday 1 is the number of viable cells per milliliter of culture asmeasured on day 1; the CVCD for day 2 is the number of viable cells permilliliter of culture as measured on day 2 plus the number of viablecells per milliliter of culture as measured on day 1; the CVCD for day 3is the number of viable cells per milliliter of culture as measured onday 3 plus the numbers of viable cells per milliliter of culturemeasured on days 1 and 2; and CVCDs for subsequent days are calculatedin a similar manner.

Results. Higher CVCDs were achieved in the presence of little or nocaffeine. Lower temperature, i.e., 36° C. rather than 37° C., and lowerlevels of caffeine resulted in higher final viability. Caffeine at 2.5mM resulted in cell death and termination of the cultures. Over the restof the concentration range tested, increased levels of caffeine resultedin increased cumulative specific productivity (Cum Qp), with the highestlevel being almost 30 μg/10⁶ cells/day. Cultures containing the highestlevels of caffeine resulting in viable cultures (2 mM), while having ahigh Cum Qp, had a low CVCD, indicating that 2 mM caffeine decreasedcell viability but increased the productivity of remaining viable cells.However, protein titers of cultures induced with 2 mM caffeine werelower than for uninduced cultures at 7 days at both temperatures.

The highest protein titers resulted at the low to intermediate levels ofcaffeine for both temperatures. The highest day 7 titer was observed inthe culture grown at 36° C. in the presence of 0.5 mM caffeine, and itstiter was about 124% of the titer seen in a control culture grown at 36°C. for 7 days without inducers. Day 7 titers of 36° C. cultures grown inthe presence of 1.0 mM and 1.5 mM caffeine were about 116% and 111% ofcontrol levels, respectively. The day 7 titers of cultures grown at 37°C. in the presence of 0.5 mM, 1.0 mM, and 1.5 mM caffeine were about110%, 112%, and 109%, respectively, of the 37° C. no inducer controlculture. Together, these data indicate that induction of cell line #60was more effective at 36° C. than it was at 37° C. Day 7 titers ofcontrol cultures without inducers grown at 36° C. and 37° C. werecomparable. Thus, as in Example 3, the lowest concentration of caffeinetested led to the highest protein titers at 36° C., suggesting thepossibility that even lower concentrations might produce equal or highertiters.

In summary, induction with caffeine increased specific productivity andtiter at both 36° C. and 37° C. Titers were modestly higher at 36° C.than at 37° C., despite lower Cum Qp values because of higher CVCDs andviability at the lower temperature. Based on cell performance andproductivity, caffeine can be used to induce production from this cellline.

EXAMPLE 5 Induction Effects for Compounds Related to Caffeine in CellLine #9

Since the above experiments showed that caffeine as an inducing agentincreased titers of recombinant polypeptide between about 9% and about67%, additional experiments were performed with other xanthinederivatives to test their inducing ability. Based upon the structure ofxanthine, a variety of compounds were modeled and chosen for testing.These include 3-isobutyl-1-methylxanthine, theophylline, theobromine,pentoxifylline, and aminophylline, the structures of which areillustrated below. TABLE 2 Xanthine Derivatives

Compound X Y Z caffeine methyl methyl methyl 3-isobutyl-1- methylisobutyl hydrogen methylxanthine (IBX) theophylline methyl methylhydrogen theobromine hydrogen methyl methyl pentoxifylline 5-oxyhexylmethyl methyl

Aminophylline is theophylline compound with 1,2-ethylenediamine (2:1)dihydrate.

These xanthine derivatives, including some combinations, were tested oncell line #9 in a shake-flask format (20 ml in 125 ml shake flasks) asdescribed above for Examples 1 and 2. To dissolve3-isobutyl-1-methylxanthine (IBX), it was solubilized in water heated toalmost the boiling point, and quickly added to the flasks before itprecipitated. Alternatively, IBX was dissolved in DMF. The inductionphase of the cell culture was carried out for 6 days at 31° C., andsamples were removed for analysis at 3 day and 6 day timepoints. Theprotein titers from each shake flask are shown in Table 3. TABLE 3Recombinant Polypeptide Titer Under Various Inducing Conditions Titer(μg/ml) Titer (μg/ml) Condition Day 3 Day 6 0.6 mM caffeine + 0.5 mMbutyrate 120 280 0.1 mM theobromine  90 270 0.5 mM theobromine 100 260 1mM theobromine 100 260 0.1 mM aminophylline 110 260 0.5 mM aminophylline120 250 1 mM aminophylline 100 200 0.1 mM pentoxyphylline 110 320 0.5 mMpentoxyphylline 130 380 1 mM pentoxyphylline 130 400 0.3% DMF + 0.5 mMIBX not determined 340 0.3% DMF 120 330 0.5 mM IBX 150 420 0.5 mM IBX +0.6 mM caffeine 130 370 0.1 mM IBX + 0.6 mM caffeine 140 390 0.1 mMIBX + 0.6 mM 130 280 caffeine + 0.5 mM butyrate 0.1 mM IBX + 0.6 mM 150390 caffeine + 0.3% DMF 0.2 mM caffeine 150 400 0.6 mM caffeine 120 3200.5 mM butyrate 100 220 NO INDUCER 100 290Several conclusions can be made from this data. Production from theflask induced with 0.5 mM IBX was even better than caffeine, and the 3flasks containing pentoxyphylline also gave promising results. Titers ofpentoxyphylline-induced cultures increased with increasing dose and werehigher than the tier of the no inducer control culture. Additionally,some combinations of different xanthine derivatives, as well asdifferent xanthine derivatives with other inducing agents (e.g.,butyrate and/or DMF) yielded protein titers above control levels.Theobromine and aminophylline did not induce protein titers above thatseen in the no inducer control culture. The highest protein titersobtained when caffeine was used as an inducer were obtained at thelowest concentration tested, that is, 0.2 mM caffeine. As explainedabove, such a result leaves open the possibility that even lowerconcentrations of caffeine may be effective. Finally, unlike in Example2 (FIG. 2), butyrate does not induce increased protein titer over thatseen in a culture with no inducer.

EXAMPLE 6 Induction of Cell Line #60 by Various Inducing Agents at 37°C.

This experiment was done in shaken Erlenmeyer flasks as described abovein Examples 1 and 2, except that the flasks were incubated for 5 days at37° C., rather than at 31° C., and cell line #60 was used. As a control,one flask without inducers was grown at 31° C. The titer of recombinantpolypeptide in the medium was assayed after 5 days. Xanthine derivativestested included caffeine (at 0.5 mM), theobromine (at 0.1 mM, 0.5 mM,and 1.0 mM), 3-isobutyl-1-methylxanthine (IBX, at 0.05 mM, 0.1 mM, and0.15 mM), and pentoxyphylline (at 0.1 mM, 0.5 mM, and 1.0 mM). Inaddition, butyrate, some combinations of inducers, and the non-xanthinecompound papaverine were tested.

The 31° C. control culture yielded low protein titers compared to the37° C. control culture, probably due to the preference of cell line #60for higher temperatures. Theobromine at a concentration of 0.11 mMincreased protein titer over that seen in the 37° C. control culture,but was counterproductive at higher concentrations (0.5 mM and 1.0 mM).Neither caffeine, IBX, or pentoxyphylline increased protein titers abovethat seen in a control culture with no inducers. Protein titer wasinversely proportional to theobromine, IBX, and pentoxyphyllineconcentrations in the ranges tested. Interestingly, cell line #9 (Table3, Example 5) showed increased protein titers with increasingpentoxyphylline concentrations within this same range, highlighting thevariability in the responses of different cell lines incubated atdifferent temperatures to inducing agents. As in other experiments (seeExample 3), 0.5 mM caffeine appears to be a better inducer than 0.5 mMbutyrate for cell line #60, although both failed to increase proteintiter over that seen in the control culture with no inducing agent inthis experiment. In a previous experiment, caffeine had induced slightlyhigher protein production than that seen in a control culture at 37° C.at day 7 (about 110% of the titer seen in the control culture), althougha greater induction was observed at 36° C. Example 4. The failure ofcaffeine to induce increased protein production in this experiment maybe explained by a variety of factors such as experimental variability,the small size of the positive effect at 37° C. in cell line #60, and/orthe possibility that 0.5 mM may not be an optimum caffeine concentrationfor induction of cell line #60 at 37° C.

EXAMPLE 7 Production of RANK:Fc in the Presence of Varying Amounts ofHMBA

Nucleic acids encoding RANK:Fc inserted into a suitable vector (asdescribed in International Application WO 01/36637) were introduced intoCHO cells. About 2 million cells from a stably transformed linepropagated at 37° C. were inoculated into 20 milliliters of medium at31° C., either without HMBA or in the presence of varying concentrationsof HMBA, as indicated in Table 4. Cells were grown for a total of 5 daysin shaker flasks. Thereafter, all medium was harvested. The number ofcells present in the culture was determined by staining with trypan blueand counting the cells in a hemocytometer. The titer of RANK:Fc permilliliter of harvested medium was determined by purifying RANK:Fc byProtein A high performance liquid chromatography (HPLC) and subsequentlymeasuring absorbance at 280 nanometers. An average number of cells inthe culture was calculated by averaging the starting and ending cellnumbers. Specific productivity was determined from the total number ofmicrograms of RANK:Fc produced, an average cell number (calculated asdescribed above), and the number of days of growth. Data from thisexperiment are shown in Table 4. TABLE 4 Effects of VaryingConcentrations of HMBA on Protein Titer and Specific Productivity HMBASpecific Titer of concentration productivity RANK: Fc (mM) (μg/10⁶cells/day) (μg/ml) 0 17.1 272 0.1 19.1 243 0.5 19.5 347 2.0 23.9 444

These data indicate that the addition of HMBA at concentrations of 0.5or 2.0 mM had positive effects on polypeptide production and specificproductivity.

EXAMPLE 8 Production of RANK:Fc in the Presence of HMBA, Caffeine,and/or Butyric Acid

Nucleic acids encoding RANK:Fc inserted into a suitable vector (asdescribed in International Application WO 01/36637) were introduced intoCHO cells. About 2 million cells from a stably transformed linepropagated at 37° C. were inoculated into 20 milliliters of serum-freemedium at 31° C. without inducers or in the presence of of HMBA and/orcaffeine and/or butyric acid, as indicated in Table 5. Cells were grownfor a total of 5 days in a shaker flask. Thereafter, all medium washarvested. The number of cells present in the culture, the titer ofRANK:Fc per milliliter of harvested medium, and specific productivitywere determined as described above in Example 7. Data from thisexperiment are shown in Table 5. TABLE 5 Effects of Caffeine, HMBA, andButyric Acid Singly and in Combination on Protein Titer and SpecificProductivity Specific Titer of productivity RANK: Fc Inducer (μg/10⁶cells/day) (μg/ml) None 14.8 261 HMBA (2 mM) 22.8 410 Caffeine (1 mM)23.6 362 Butyric acid (0.5 mM) 31.6 476 HMBA (2 mM) + Caffeine 46.7 553(1 mM) + butyric acid (0.5 mM)

These data indicate that the addition either caffeine (at 1 mM), butyricacid (at 0.5 mM), or HMBA (at 2 mM) had positive effects on bothpolypeptide production and specific productivity and that thecombination of butyric acid, caffeine, and HMBA (at the concentrationsmentioned above) had greater positive effects than any of thesecompounds alone.

EXAMPLE 9 Production of Type H IL-1 Receptor in the Presence of HMBA ina Bioreactor

Nucleic acids encoding a type II IL-1 receptor inserted into a suitablevector were introduced into CHO cells. About 500 thousand cells from astably transformed line were inoculated into a one liter of serum-freemedium in a bioreactor. Cells were grown for two days at 37° C.Thereafter, cells were shifted to 31° C., either without HMBA or in thepresence of 2 mM HMBA, and grown for 12 more days. Thereafter, allmedium was harvested. The titer of type II IL-1 receptor per milliliterof harvested medium was determined by purification by reverse phase HPLCfollowed by the measurement of absorbance at 280 nanometers. Data fromthis experiment are shown in Table 6 as a percentage of the average ofthe protein titers obtained from the two samples without HMBA rounded tothe nearest whole number. TABLE 6 Effects of 2 mM HMBA on Protein TiterRelative Titer of type II IL-1 Receptor (percent of average of Inducersamples without HMBA) None  99% None 101% HMBA (2 mM) 120% HMBA (2 mM)125%

These data show that bioreactor cultures shifted to 31° C. after aninitial 37° C. growth phase produced more type II IL-1 receptor if HMBAwas added at the time of temperature shift than if it wasn't. These datafurther suggest that the invention can be useful for producing a varietyof polypeptides in a variety of cell lines and that the mechanics of howthe cells are grown, for example, in a shaker flask versus in abioreactor, are not critical.

EXAMPLE 10 Production of an Antibody Against Murine IL-4 Receptor in CHOCells

The experiment described below tests the effects of using either sodiumbutyrate or HMBA as an inducer in still another cell line at varioustemperatures.

Nucleic acids encoding an antibody against a murine IL-4 receptorinserted into a suitable vector were introduced into CHO cells. Abouttwo million cells from a stably transformed line propagated at 37° C.were inoculated into 20 milliliters of medium at the temperaturesindicated in FIG. 4 and in the presence or absence of HMBA (2 mM) orsodium butyrate (0.5 mM), as indicated in FIG. 4. Cells were grown for amaximum of 14 days in a shaker flask. Aliquots were removed at the timesindicated in FIG. 4, and the titer of the antibody (in micrograms permilliliter of harvested medium) was determined by enzyme-linkedimmunosorbent assay (ELISA), a method well known in the art. See e.g.Reen (1994), Enzyme-Linked Immunosorbent Assay (ELISA), in Basic Proteinand Peptide Protocols, Methods Mol. Biol. 32:461-466. The results areshown in FIG. 4. These data indicate that growth at 31° C. resulted inthe production of more antibody for a longer time than growth at either34° C. or 37° C. when medium was harvested at 7 days or later. Thesedata also indicate that both HMBA and sodium butyrate, individually,enhanced production of the antibody and that HMBA did so to a greaterextent than did sodium butyrate at 31° C.

EXAMPLE 11 Production of TNFR:Fc in CHO Cells

Nucleic acids encoding human TNFR:Fc in a suitable vector wereintroduced into CHO cells. About 3±0.5×10⁶ cells from a stablytransformed cell line propagated at 37° C. were introduced into each ofthree 1 liter bioreactors and cultured at 32.5° C. in an enriched,serum-free medium. Sodium butyrate (0.5 mM) was added to all threecultures, and HMBA (2 mM) was added to two of the cultures (“day1+HMBA”) one day after the shift to 32.5° C. Cells were incubated for atotal of 11 days at 32.5° C. Medium was harvested, and protein titer wasdetermined by measuring optical density at 280 nanometers following aprepurification using Protein A POROS® Perfusion Chromatography™(Applied Biosystems, Foster City, Calif., USA). These results are shownin Table 7 as a percentage of the titer obtained from the sample with noHMBA (“day 1”) rounded to the nearest whole number. TABLE 7 Effects ofthe Timing of Addition of Inducers Relative Titer TNFR: Fc (percent ofthe day 0 titer) day 1 100% day 1 + HMBA 131% day 1 + HMBA 110%

These data indicate that the addition of HMBA increased the titer ofTNFR:Fc produced by these cultures when added one day after atemperature shift to 32.5° C. These data, together with the data inprevious examples, indicate that addition of HMBA can increase proteintiter when it is added at the time of or after a shift to a lowertemperature.

The foregoing description of specific embodiments reveals the generalnature of the invention so that others can readily modify and/or adaptsuch embodiments for various applications without departing from thegeneric concepts presented herein. Any such adaptions or modificationsare intended to be embraced within the meaning and range of equivalentsof the disclosed embodiments. Phraseology and terminology employedherein are for the purpose of description and not of limitation. Allreferences cited herein are hereby incorporated by reference in theirentirety.

1. A method for producing a recombinant polypeptide comprising culturinga mammalian cell line that can produce the polypeptide at a temperatureless than 37° C. in a medium comprising a hybrid polar compound.
 2. Themethod of claim 1, wherein the hybrid polar compound is hexamethylenebisacetamide at a concentration from about 0.1 millimolar to about 20millimolar.
 3. The method of claim 2, wherein the hexamethylenebisacetamide is at a concentration from about 0.1 millimolar to about 5millimolar.
 4. The method of claim 1, wherein the mammalian cell line iscultured at a temperature from about 29° C. to about 36° C.
 5. Themethod of claim 4, wherein the mammalian cell line is cultured at atemperature from about 30° C. to about 33° C.
 6. The method of claim 1,wherein the medium is serum free.
 7. The method of claim 1, wherein themedium further comprises a xanthine derivative.
 8. The method of claim1, wherein the mammalian cell line is a CHO cell line.
 9. The method ofclaim 1, wherein the recombinant polypeptide is a secreted polypeptideand wherein the method further comprises recovering the recombinantpolypeptide from the medium.
 10. A method for producing a polypeptidecomprising culturing a CHO cell line that can produce the polypeptide ata temperature of less than 37° C. in a medium comprising a hybrid polarcompound.
 11. The method of claim 10, wherein the hybrid polar compoundis hexamethylene bisacetamide at a concentration from about 0.1millimolar to about 20 millimolar.
 12. The method of claim 11, whereinthe hexamethylene bisacetamide in the medium is present at aconcentration from about 0.1 millimolar to about 5 millimolar.
 13. Themethod of claim 10, wherein the medium is serum free.
 14. The method ofclaim 10, wherein the medium further comprises a xanthine derivative.15. The method of claim 10, wherein the polypeptide is a secretedpolypeptide and wherein the method further comprises recovering thepolypeptide from the medium.
 16. The method of claim 10, wherein thepolypeptide is a recombinant polypeptide or an antibody.
 17. A methodfor producing a polypeptide comprising: culturing a mammalian cell linein a growth phase followed by a production phase, wherein the productionphase occurs at a temperature of less than 37° C.; and adding to theculture medium during the production phase a xanthine derivative;wherein the mammalian cell line is selected from the group consisting ofa mammalian cell line that has been genetically engineered to producethe polypeptide and a hybridoma cell line that produces an antibody. 18.The method of claim 17, wherein the xanthine derivative is caffeine at aconcentration from about 0.01 millimolar to about 3.0 millimolar. 19.The method of claim 17, wherein the polypeptide is a recombinant fusionpolypeptide or a human or humanized antibody.
 20. The method of claim17, wherein the production phase occurs at a temperature from about 29°C. to about 36° C.
 21. The method of claim 17, wherein the mammaliancell line is a CHO cell line.
 22. The method of claim 17, wherein themedium used during the production phase is serum free.
 23. A method forproducing a recombinant polypeptide comprising: culturing a CHO cellline that has been genetically engineered to produce the recombinantpolypeptide; and adding to the culture medium at least one xanthinederivative selected from the group consisting of theobromine andcaffeine.
 24. The method of claim 23, wherein the recombinantpolypeptide is a fusion polypeptide or a human or humanized antibody.25. The method of claim 23, wherein the concentration of each xanthinederivative added to the culture medium is from about 0.001 millimolar toabout 3 millimolar.
 26. The method of claim 23, wherein the xanthinederivative is caffeine.
 27. The method of claim 23, wherein the CHO cellline is cultured at a temperature from about 29° C. to about 36° C.